The long term goals of this project are to utilize gene transfer technologies to deliver drug resistant genes into hematopoietic stem and progenitor cells, thus allowing relevant chemotherapeutic agents to be administered to patients with lessened myelosuppression, and in higher doses than would be otherwise tolerated. This project bring together the expertise of the PI, who has developed mutant forms of the enzymes dihydrofolate reductase (DHFR) and thymidylate synthase (TS), targets for methotrexate (MTX) and 5-fluoropyrimidines and the Co-PI, with expertise in stem cell biology, aided by the retroviral core and the imaging core, to accomplish its goals. Based on progress during the previous grant period we will initiate a pilot trial in patients with lymphoma, using a retroviral vector containing a fusion double mutant DHFR and cytidine deaminase cDNA to transduce CD34+ cells. The goal of this study is to determine if significant levels of the fusion protein are expressed in hematopoietic cells, and if subsequent treatment with escalating doses of MTX and cytosine arabinoside increases the percentage of blood cells that express this fusion protein. Additional aims are to generate and evaluate a mutant DHFR TS fusion protein, and to generate and study retroviral constructs containing MRP-1, a multi-drug resistance associated protein. Generation of these constructs will broaden the use of this use of gene therapy to encompass other tumor types (breast, bladder), in which drugs are used where toxicity may be ameliorated by successful stem cell transfection and expression of drug resistant genes. Efforts will continue to further optimize human stem cell transduction, and to utilize the NOD-SCID mouse for this purpose. Gene transfer into non-traditional stem cell populations, and the role of stem cell proliferative senescence due to telomere shortening as a limitation of successful long term gene transfer into stem cells will also be studied.